PROJECT SUMMARY/ABSTRACT Antibiotic resistant infections are a dangerous, worldwide health problem. Chief among these pathogens is Mycobacterium tuberculosis (Mtb), which causes an estimated 1.5 million deaths a year. The emergence of drug-resistant Mtb strains, which constitute 20% of previously treated tuberculosis (TB) cases, has exacerbated this already alarming epidemic. The inadequacies of present TB therapies demand the discovery of new agents with unique mechanisms of action to treat Mtb infection. Towards this end, we have discovered and developed a new family of compounds (termed Mycobacterial Tolerance Inhibitors, MTIs) that potently sensitize Mtb to stresses encountered during infection as well as to the frontline antibiotic isoniazid (INH). In addition, we show that combining MTIs with INH blocks the selection for INH-resistance and restores INH activity in otherwise INH-resistant Mtb isolates. To the best of our knowledge, MTIs are the first report of compounds that block INH resistance and re-sensitize INH-resistant bacteria to INH. INH is included in the standard of care (SOC) regimens for both prophylactic treatment of latent TB as well as treatment of active TB. Unfortunately, the increase in INH-resistant cases of TB is threatening the relevance of this antibiotic, which would generate a large gap in our treatment options. MTIs represent an innovative strategy for combating TB drug resistance. In preliminary mechanistic work, we show that MTIs induce expression profiles and changes in physiology that are indicative of dysregulation of respiration. Therefore, we hypothesize that MTIs modulate respiration in Mtb, leading to sensitization to stresses associated with respiration, potentiation of INH activity, and re-sensitization of INH-resistant mutants to INH, which will all improve the efficacy of antibiotic therapy and immune responses during infection. We have demonstrated that MTIs have pharmacokinetic (PK) properties suitable for oral dosing, further supporting their promise for translation to treatments in humans. Our short-term objectives are to demonstrate preclinical proof-of-concept for MTIs to combat Mtb infection, optimize the current lead MTIs for translation to a therapeutic, and reveal new insights into pathways of drug tolerance and resistance. Our long-term objective is to develop a new orally available antibiotic that improves the current SOC regimens for patients with drug-sensitive and resistant TB. Using our combined expertise in chemistry and Mtb biology, we will achieve our objectives by addressing the following independent aims: 1) Optimize MTIs through structure-activity relationships (SAR) and structure-property relationships (SPR) studies. 2) Dissect the exact mode of action and the detailed mechanisms by which MTIs impact INH and stress sensitivity. 3) Determine the activity of MTIs during Mtb infection. Successful completion of these aims will result in the development of an innovative therapeutic strategy to combat drug tolerance and drug resistance.